Isolation and Culture of Neonatal Murine Primary Cardiomyocytes

Ravi, V.; Jain, Aditi; Taneja, Arushi; Chatterjee, Kaushik; Sundaresan, Nagalingam R.

doi:10.1002/cpz1.196

Cited by 24 publications

(12 citation statements)

References 63 publications

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“…Although cultured cardiac fibroblasts cannot completely mimic the in vitro phenotype, they prove to be an advantageous model in several aspects for both physiological and pathophysiological studies. For example, they can be selectively manipulated with numerous treatments without interference from cardiomyocytes as they are cultured as a distinct homogenous population ( Ravi et al, 2021 ). These cells can be used for various genetic and/or drug screening purposes, which may be completed in comparatively less time than in vitro studies.…”

Section: Methodsmentioning

confidence: 99%

Isolation and Culture of Primary Fibroblasts from Neonatal Murine Hearts to Study Cardiac Fibrosis

Kumar¹,

Nagesh²,

Ramasubbu³

et al. 2023

BIO-PROTOCOL

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Cardiac fibroblasts are one of the major constituents of a healthy heart. Cultured cardiac fibroblasts are a crucial resource for conducting studies on cardiac fibrosis. The existing methods for culturing cardiac fibroblasts involve complicated steps and require special reagents and instruments. The major problems faced with primary cardiac fibroblast culture are the low yield and viability of the cultured cells and contamination with other heart cell types, including cardiomyocytes, endothelial cells, and immune cells. Numerous parameters, including the quality of the reagents used for the culture, conditions maintained during digestion of the cardiac tissue, composition of the digestion mixture used, and age of the pups used for culture determine the yield and purity of the cultured cardiac fibroblasts. The present study describes a detailed and simplified protocol to isolate and culture primary cardiac fibroblasts from neonatal murine pups. We demonstrate the transdifferentiation of fibroblasts into myofibroblasts through transforming growth factor (TGF)-β1 treatment, representing the changes in fibroblasts during cardiac fibrosis. These cells can be used to study the various aspects of cardiac fibrosis, inflammation, fibroblast proliferation, and growth.

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Section: Methodsmentioning

confidence: 99%

Isolation and Culture of Primary Fibroblasts from Neonatal Murine Hearts to Study Cardiac Fibrosis

Kumar¹,

Nagesh²,

Ramasubbu³

et al. 2023

BIO-PROTOCOL

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“…NRCM cultures were prepared using a modified protocol, as previously described [ 35 ]. NRCMs were obtained from 1-day-old male Wistar rats, which were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences (SLACCAS).…”

Section: Methodsmentioning

confidence: 99%

Trans-cinnamaldehyde protects against phenylephrine-induced cardiomyocyte hypertrophy through the CaMKII/ERK pathway

Qian

Tian

Wang

et al. 2022

BMC Complement Med Ther

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Background Trans-cinnamaldehyde (TCA) is one of the main pharmaceutical ingredients of Cinnamomum cassia Presl, which has been shown to have therapeutic effects on a variety of cardiovascular diseases. This study was carried out to characterize and reveal the underlying mechanisms of the protective effects of TCA against cardiac hypertrophy. Methods We used phenylephrine (PE) to induce cardiac hypertrophy and treated with TCA in vivo and in vitro. In neonatal rat cardiomyocytes (NRCMs), RNA sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were carried out to identify potential pathways of TCA. Then, the phosphorylation and nuclear localization of calcium/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-related kinase (ERK) were detected. In adult mouse cardiomyocytes (AMCMs), calcium transients, calcium sparks, sarcomere shortening and the phosphorylation of several key proteins for calcium handling were evaluated. For mouse in vivo experiments, cardiac hypertrophy was evaluated by assessing morphological changes, echocardiographic parameters, and the expression of hypertrophic genes and proteins. Results TCA suppressed PE-induced cardiac hypertrophy and the phosphorylation and nuclear localization of CaMKII and ERK in NRCMs. Our data also demonstrate that TCA blocked the hyperphosphorylation of ryanodine receptor type 2 (RyR2) and phospholamban (PLN) and restored Ca2+ handling and sarcomere shortening in AMCMs. Moreover, our data revealed that TCA alleviated PE-induced cardiac hypertrophy in adult mice and downregulated the phosphorylation of CaMKII and ERK. Conclusion TCA has a protective effect against PE-induced cardiac hypertrophy that may be associated with the inhibition of the CaMKII/ERK pathway.

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“…The detailed protocol for culturing primary cardiomyocytes has been adapted from Jain et al (2017) and Ravi et al (2021).…”

Section: Cell Seeding and Experimental Treatment Timing: 3-4 Daysmentioning

confidence: 99%

“…The detailed protocol for culturing primary cardiomyocytes has been adapted from Jain et al (2017) and Ravi et al (2021). The following additional steps are to be followed for pre-treatment of culture plates before seeding primary cardiomyocytes for confocal microscopy.…”

Section: Preparation Of Cell Culture Plates For Confocal Microscopymentioning

confidence: 99%

Microscopy‐Based Assessment of Fatty Acid Uptake and Lipid Accumulation in Cultured Cells

et al. 2022

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The heart relies predominantly on the use of fatty acids to derive energy. Metabolic disorders such as obesity, insulin resistance, and diabetes pose a major risk factor for the development of heart failure. Dysregulation of lipid metabolism observed in these diseases manifests as cardiac lipotoxicity, and is associated with cardiac dysfunction. The alarming rise in the incidence of these metabolic disorders warrants the need for tools to investigate the underlying molecular mechanisms. In this article, we describe a confocal microscopybased approach to monitor fatty acid uptake and lipid accumulation in vitro, in neonatal murine cardiomyocytes and H9c2 cells. The protocol for assessment of fatty acid uptake relies on the use of BODIPY FL C 12 TM to study the kinetics of fatty acid uptake via real-time imaging of fatty acid uptake in live cells. Importantly, it circumvents the need for radioactive labeling of fatty acids to evaluate their uptake. Similarly, the protocol for assessment of lipid accumulation relies on the use of BODIPY TM 493/503 to stain the cytosolic neutral lipid population in fixed cells. We couple these confocal microscopy-based approaches with fluorescence intensity analysis using FIJI to quantify fatty acid uptake and lipid accumulation in vitro.

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Isolation and Culture of Neonatal Murine Primary Cardiomyocytes

Cited by 24 publications

References 63 publications

Isolation and Culture of Primary Fibroblasts from Neonatal Murine Hearts to Study Cardiac Fibrosis

Isolation and Culture of Primary Fibroblasts from Neonatal Murine Hearts to Study Cardiac Fibrosis

Trans-cinnamaldehyde protects against phenylephrine-induced cardiomyocyte hypertrophy through the CaMKII/ERK pathway

Microscopy‐Based Assessment of Fatty Acid Uptake and Lipid Accumulation in Cultured Cells

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